Abstract
Background: The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for de novo assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.
Highlights
With the development of robust second generation bioethanol processes, next to the use of highly engineered Saccharomyces cerevisiae strains[1,2], non-classical ethanologenic yeasts are being considered as production organisms[3,4]
In our previous studies we have identified a novel ethanologenic yeast, Wickerhamomyces anomala, as a potential candidate[3]
A further screen for alternative yeast species was initiated (Punt and Omer, unpublished study) Here we describe the isolation and genomic characterization of one of these new isolates, which was typed as Candida vartiovaarae based on ribosomal RNA analysis
Summary
Keywords Nanopore sequencing, de novo genome assembly, wild type yeasts, ethanologenic, Candida, Cyberlindera. Hans Jansen generated new data and visualized mitochondrial coverage for all used chemistries. Michael Liem performed all assemblies, assembly correction and assembly comparison, gene prediction analysis and contig comparisons including all rewriting of the manuscript and generated and/or edited Figure 1, Figure 3–Figure 6. Data characteristic, such as number of reads, number of bases, coverage and GC-content, are added to the previous table
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